Synaptopodin

The procedure and protocol for IHC and IF staining have been described in our previous reports [27 (link)–32 (link)]. For IHC and IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies specifically against zonula occludens-1 (ZO-1) (1: 200, Abcam), kidney injury molecule (KIM)-1 (1: 400, Novus), synaptopodin (1:500, Santa Cruz) and alpha-smooth muscle actin (α-SMA) (A2547, 1: 500, Sigma-Aldrich), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of kidney specimen and quadriceps muscle from each rat were analyzed. For quantification, three random chosen HPFs (200 × or 400 × for IHC and IF studies) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.
An IF-based scoring system was adopted for semi-quantitative analysis of KIM-1 in the kidney as a percentage of positive cells in a blinded fashion (score of positively stained cell for these biomarkers as: 0 = negative staining; 1 = < 15%; 2 = 16–25%; 3 = 26–50%; 4 = 51–75%; 5 = 76–100% per HPF).

At P21 and 24 weeks, podocytes were counted in 20 whole glomeruli in a single 800‐μm slice from each mouse podocytes were identified by their nuclear expression of p57 (sc‐8298; Santa Cruz Biotechnology, Santa Cruz, CA) and cytoplasmic expression of synaptopodin (sc‐21,537; Santa Cruz Biotechnology). Following immunofluorescence labeling kidney slices were cleared using ethyl cinnamate (ECi; 99% concentration, product number 112372; Sigma‐Aldrich) and then imaged using a Leica SP8 Multiphoton Microscope fitted with a Leica 20× BABB objective lens. Twenty glomeruli per kidney were imaged over the z‐axis at 1‐μm intervals to obtain a stack of optical sections through each whole glomerulus.

HK-2 cells (a human renal proximal tubular epithelial cell line) were purchased from the Korean cell line bank (KCLB®, Seoul, South Korea) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ under 5% CO₂ in a humidified incubator.
Conditionally human immortalized podocytes (AB8/13 cell line) were generously provided by Professor Moin Saleem (University of Bristol, Southmead Hospital, Bristol, UK) [34 (link)]. These cells were grown in RPMI-1640 supplemented with 10% FBS, 1% penicillin/streptomycin and 1% insulin-transferrin-selenium supplement (ITS-G; Gibco, Grand Island, NY). The cells were propagated at 33℃ until reaching 80% confluence and were allowed to differentiate at 38℃ with 40–50% confluence for 14 days. Differentiation was assessed by immunofluorescence (IF) staining with podocin (Sigma Aldrich; St. Louis, MO), nephrin (Abcam, Cambridge, MA) and synaptopodin (Santa Cruz Biotechnology, Santa Cruz, CA) using a confocal laser-scanning microscope (LSM710; Carl Zeiss, Jena, Germany) (Additional file 1: Fig. S2A, B).